: The long-term goals of this research are to determine the mechanisms by which hepatocytes secrete lipoproteins and apolipoproteins. Hepatocytes secrete most of the plasma lipoproteins, and these same cells are the principal site of their uptake and catabolism. This research will focus on the secretory limb of hepatocyte lipoprotein metabolism, investigating five goals. The first is to thoroughly characterize nascent very low density lipoproteins (VLDL) isolated from the Golgi apparatus. The second is to determine the mechanism(s) by which nascent VLDL contribute to the formation of plasma high density lipoproteins (HDL). Previous research on these two questions has been difficult to interpret because the putative Golgi fractions used as a source of nascent lipoproteins were contaminated by endosomes containing remnant lipoproteins. Recently, modifications of a method for isolating intact Golgi apparatus have eliminated endosomal contamination. The properties of nascent VLDL isolated from this intact Golgi fraction are strikingly different from previous findings and provide the opportunity to ask several fundamental questions: 1) Do all nascent Golgi VLDL contain apolipoprotein (apo) B and apo E? 2) Are any of the apolipoproteins found in blood plasma secreted independently of apo B-containing particles? 3) By what mechanisms do the surface lipids and apolipoproteins of nascent Golgi VLDL contribute to the formation of plasma HDL? The third goal is to investigate very early events of apo B lipidation in the rough endoplasmic reticulum (RER). Because standard microsomal preparations are considered inadequate, a novel technique was developed for isolating very large amounts of highly pure and structurally "intact" RER fractions in less than two hours, which have 20-fold more glucosyl transferase activity than the parent live homogenate. Basic questions about early events of nascent VLDL assembly can now be asked: 1) Is apo B in the RER bound to the membrane? 2) Is apo B in the RER lipidated and if so, what lipids are first added? 3) Are cholesteryl esters produced by RER acyl-CoA cholesterol acyl transferase (ACAT) involved in apo B translocation across the RER membrane? 4) Are cholesteryl esters necessary for VLDL assembly? 5) When and how are triglycerides incorporated into apo B-containing nascent VLDL particles? The fourth goal is to investigate the hypothesis that the low density lipoprotein receptor-related protein (LRP) and apo B interact in the assembly and intracellular transport of nascent VLDL. This will be investigated by immunogold double labeling localization of cryothin sections and by binding and cross-linking studies in intact Golgi and RER fractions. A fifth goal is to generate an antigenic map of the distribution of the major plasma lipoprotein-related macromolecules in rat hepatocytes by immunogold labeling of cryothin sections. The correlation of the ultrastructural distribution of lipoprotein-related antigens with the subcellular biochemical research will contribute to understanding of nascent VLDL assembly and secretion. Answers to these basic questions may indicate ways in which VLDL assembly and secretion can be regulated as a means of controlling the levels of atherogenic and antiatherogenic lipoproteins.